[Background] Actinobacteria are a treasure trove of natural products, and 70% of the natural antibiotics currently used in clinics are derived from secondary metabolites of actinomycetes. With the increasing resistance of bacteria to traditional antibiotics, how to efficiently screen new active actinomycete resources from natural habitats and discover new antibiotics has become an important challenge for microbiologists. [Objective] However, screening active actinomycetes by traditional methods not only takes time and effort, consumes large amounts of reagent consumables, but also has a very limited screening throughput, making it difficult to analyze the complex microbial community in natural samples as a whole. This study proposes a new strategy based on micro-well plate droplet array culture, which can screen antibacterial Actinobacteria with high throughput. The study analyzed the culture characteristics and screening conditions of model actinomycetes in micro-droplets, which laid the foundation for the establishment of an ultra-high-throughput bioactive Actinobacteria screening platform based on droplet array technology. [Methods] Our new solution is to use interfacial micropipetting (IMP) technology to miniaturize the traditional multi-well high-throughput screening system to 1 μL level, and generate microliter culture droplet arrays in oil-filled micro-well plates (96-well plates), each microdroplet encapsulates an actinobacterial spore or hyphae. After a short period of cultivation, the actinobacteria complete mycelial differentiation and secretion of secondary metabolites in the microdroplets. At this time, the indicator bacteria with fluorescent markers were added through the second step of IMP and the fusion of the droplets, the active target strains were located through the antagonistic screening of the whole bacteria, and the activity spectrum was converted into quantitative fluorescence values. [Results] the model actinobacteria test was found that the mycelium can reach the optimal culture state in the microdroplets and accumulate enough biomass and metabolites, which have obvious inhibitory effects on the fluorescent indicator bacteria. [Conclusion] By establishing the above-mentioned high-throughput screening technology based on microplate droplet arrays, we can quickly screen strains with antibacterial activity from the single-cell level, which significantly saves screening costs and improves screening throughput, which provides a starting point for the discovery and study of novel functional natural products.